A number of virus and non-virus methods have been developed to introduce genes into cultured cells or biological tissues for the purposes of gene function analysis, gene therapy, and similar applications (Mulligan, Science, 260, 926 to 932, 1993; and Ledley, Human Gene Therapy, Vol. 6, 1129 to 1144, 1995). Virus methods are most effective for the delivery of genes into cells. However, virus vectors may raise problems due to the co-introduction of gene elements essential for parent genes derived from the parent virus, expression of virus genes, an immunogenicity, and the like. On the other hand, a liposome method, which is a non-virus method, has a lower level of cytotoxicity and immunogenicity than virus methods, but also tends to have a lower level of gene introduction efficiency into biological tissues that that of virus vectors.
Hemagglutinating virus of Japan (HVJ) was first reported as fusing Ehrlich tumor cells (Okada, Biken Journal, 1, 103-110, 1958), then the mechanism of its ability to fuse cell membranes (hereinafter referred to as “fusion activity”) was clarified and the possible use of it as a gene introduction vector has been studied. It is known that HVJ has a high level of immunogenicity, and particularly induces Cytotoxic T lymphocyte (CTL) when a large amount of NP protein is produced (Cole G. A. et al., Journal of Immunology, 158, 4301 to 4309, 1997). It is also likely that HVJ inhibits protein synthesis in hosts. To avoid these problems, a technique was devised in which a liposome including a gene or protein is fused with HVJ which has been inactivated by ultraviolet irradiation to prepare a fusion particle (HVJ-liposome) This technique made it possible to introduce a gene non-invasively into cultured cells or organisms (U.S. Pat. No. 5,631,237; Dzau et al., Proc. Natl. Acad. Sci. USA, 93, 11421 to 11425, 1996, and Kaneda et al., Molecular Medicine Today, 5, 298 to 303, 1999). However, the technique requires preparation of two different vehicles, a liposome and a viral envelope, which complicates the technique. The fusion particle of a liposome and HVJ disadvantageously has an average diameter about 1.3 times that of HVJ and a fusion activity one-tenth that of HVJ. In addition, for conventional HVJ-based vectors, there are some tissues in which it is not possible to introduce genes, or if it is possible, it is only possible with very low efficiency.
The present inventors have provided various novel inactivated virus envelope vectors for introducing a gene or oligonucleotide into cultured cells or organisms (WO01/57204). Specifically, by packaging genes into envelopes of various envelope viruses (e.g., HVJ, etc.), whose genomes are previously inactivated, the resultant viruses can be used as vectors capable of introducing genes into cultured cells or biological tissues with simplicity and high efficiency. These viral vectors are also less toxic to cells.
The present inventors have developed a method for producing an inactivated viral envelope at an industrial scale which is inexpensive, effective and secures a good quality product, by employing the use of an alkylating agent.
In the present days, although some treatments exist for many diseases, diseases and disorders of the brain is a field where few solutions are found. Further, demand for such treatment and prevention is increasing very year.
Among encephalopathies, cerebral occlusive disease caused by atherosclerosis of the cerebral arteries or Moyamoya disease, often causes chronic hypoperfusion of the brain. Although such a condition leads not only to cerebral ischemic events, but also to neuropathological changes including dementia (Kalaria R N, Bhatti S U, Lust W D, Perry G., Ann N Y Acad Sci. 1993; 695; 190-3.; Kudo T, Takeda M, Tanimukai S, Nishimura T., Stroke. 1993; 24:259-64; discussion 265.; Kurumatani T, Kudo T, Ikura Y, Takeda M., Stroke. 1998; 29:1058-62.; and Sekhon L H, Morgan M K, Spence I, Weber N C., Stroke. 1994; 25:1022-7), an effective treatment of hypoperfusion has not yet been established.
Recently, preclinical studies have demonstrated that angiogenic growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and hepatocyte growth factor (HGF) can stimulate the development of collateral arteries in an animal brain ischemia model (Harrigan M R, Ennis S R, Masada T, Keep R. F., Neurosurgery. 2002; 50:589-98; Lyons M K, Anderson R E, Meyer F B., Brain Res. 1991; 558:315-20.; and Yoshimura S, et al., Hypertension. 2002; 39:1028-34.), a concept called therapeutic angiogenesis. The efficacy of therapeutic angiogenesis using gene transfer of angiogenic growth factors has been reported in human patients with critical limb ischemia or myocardial infarction (Baumgartner I, et al., circulation. 1998; 97:1114-23.; Losordo D W, et al., Circulation. 1998; 98:2800-4.; Symes J F, et al., Ann Thorac Surg. 1999; 68:830-6; discussion 836-7.; and Rosengart T K, et al., Circulation. 1999; 100:468-74.). Accordingly, possible therapeutic angiogenesis should be possible for the treatment of patients with cerebral ischemia, if safe and effective gene transfer methods were developed for human treatment. From this viewpoint, the current gene transfer techniques are not ideal. Gene transfer to the central nervous system (CNS) can be achieved by using various viral vectors including adeno-associated virus (AAV) (Fan D, et al., Neurosci Lett. 1998; 248:61-4), retrovirus (Franceschini I A, et al., J Neurosci Res. 2001; 65:208-19), adenovirus (Miyaguchi K, Maeda Y, Collin C, Sihag R K., Brain Res Bull. 2000; 51:195-202.) and herpes simplex virus 1 (Johnson P A, Yoshida K, Gage F H, Friedmann T., Brain Res Mol Brain Res. 1992; 12:95-102.). These vector systems have advantages and disadvantages for human gene therapy. Although these methods are efficient for in vivo gene transfer into CNS, numerous problems such as safety and production are yet to be resolved toward human gene therapy.
In order to solve these problems, we have developed and used HVJ (Hemagglutinating Virus of Japan =Sendai virus) envelope vector, which is a novel non-viral vector system, as mentioned above. This vector system was developed based on HVJ-based gene transfer of the first generation using a viral envelope and liposome (Yamada K, et al., Am J Physiol. 1996; 271:R1212-20; Kaneda Y, et al., Exp Cell Res. 1987; 173:56-69.) (HVJ liposome methods). The first generation HVJ vector has great potential with regards to transfection to the CNS of rat and primate (Yamada K. et al. ibid; Hagihara Y. et al., Gene Ther. 2000; 7:759-63.). However, there are some deficiencies in that it requires complicated procedure to prepare the vector, and has difficulties in its storage. A HVJ-envelope (HVJ-E) vector, a novel non-viral vector system, merely uses an envelope of HVJ for transfer of a foreign gene.